Bacillus anthracis, the causative agent of the highly infectious disease anthrax, belongs to the Bacillus cereus group, which also contains six other closely related species: Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis and Bacillus medusa. A variety of methods have been reported for the selective identification of B. anthracis. These include direct testing of bacterial DNA with specific probes, PCR amplification followed by an in-tube assay, PCR with subsequent electrophoretic analysis of length variation among ribosomal operons, ribotyping, amplified fragment length polymorphisms, methods of analysis using plasmid and chromosomal sequences, PCR-ELISA, on chip PCR amplification of anthrax toxin genes, detection of unique polysaccharides and other biomarkers on B. anthracis cell surface with mass spectrometry, immunological recognition of spores and vegetative cells and determination of phenotypic characteristics. The main goal of the various methods is rapid and inexpensive detection of this extremely pathogenic microorganism so that containment, destruction of the pathogens and treatment are facilitated.
Hybridization analysis of 16S rRNA is a method of microbial identification. The 16S rRNA molecule is suitable for use as a target for microbial identification and detection. Although conserved in sequence overall, the 16S rRNAs actually exhibit significant variation in some regions. These differences in 16S rRNA sequences provide the basis for the design of nucleic acid probes of varying specificity, ranging from probes targeting all living organisms, to group-specific and species-specific probes. Another advantage of using the rRNAs as a target is the fact that these molecules are naturally amplified within the cell. In general, rRNA represents about 80% of total nucleic acids in microbial cells, and thus is present in many hundreds and thousands of copies per cell. This natural amplification allows for direct detection of rRNA sequences without the need for intermediate amplification via PCR.
The main limitations of current hybridization techniques in general are that they are time consuming and limited in terms of the number of probes which can be analyzed simultaneously. Oligonucleotide microchip technology is a rapid and high throughput platform for nucleic acid hybridization reactions. Moreover, a universal mini-column (syringe-operated silica mini-column) for nucleic acid isolation, fractionation, fragmentation, fluorescent labeling, and purification, as well as an inexpensive, portable fluorescent analyzer for hybridization imaging was reported. Using the prototype mini-column, oligonucleotide microchip and portable imager, hybridization patterns from both microbial and human cells were detected in less than 60 minutes.
Current detection techniques for B. anthracis identification such as PCR, electrophoretic analysis, PCR-ELISA, and mass-spectrometry require a considerable amount of time, are expensive, and are generally limited by the number of probes analyzed. Moreover, some of these detection techniques are incapable of discriminating closely related isolates, especially isolates that are differentiated by as little as a single base change in DNA or RNA. In addition to being expensive and time-consuming, many of these methods are not portable. The present invention is designed to address many of the problems mentioned above.